Polypeptides Having Phytase Activity And Polynucleotides Encoding Same

ABSTRACT

The present invention relates to polypeptides having phytase activity. These polypeptides have an amino acid sequence which has at least 70% identity to either of three phytases derived from the bacterium  Buttiauxella , and which comprises at least one of the following amino acids at the position indicated: 119N, 120L, and/or 121E. These phytases have an improved specific activity. Additional specific amino acid substitutions are also disclosed which characterize and distinguish additional phytases of the invention having improved properties such as temperature and/or pH stability, pH activity profile, temperature activity profile, substrate profile, improved performance in animal feed in vitro or in vivo. The invention also relates to isolated polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority or the benefit under 35 U.S.C. 119 ofEuropean application no. 07101395 filed Jan. 30, 2007 and U.S.provisional application No. 60/887,242 filed Jan. 30, 2007, the contentsof which are fully incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

REFERENCE TO DEPOSITS OF BIOLOGICAL MATERIAL

This application contains a reference to deposits of biological materialwhich have been made at DSMZ (Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH) under the Budapest Treaty and assigned accessionnumbers DSM 18930, DSM 18931, and DSM 18932, which microbial depositsare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to isolated polypeptides having phytaseactivity and isolated polynucleotides encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the polynucleotides as well as methods for producingand using the polypeptides.

DESCRIPTION OF THE RELATED ART

WO 2006/043178 discloses a phytase from Buttiauxella P1-29 (deposited asNCIMB 41248) having the amino acid sequence of SEQ ID NO: 3 in WO2006/043178, as well as certain variants thereof. The sequence of aButtiauxella wildtype phytase and a number of variants thereof have beensubmitted to the GENESEQP database with the following accession numbers,AEH25051, AEH25056, AEH25057, AEH25058, AEH25059, AEH225060, AEH225061AEH25062, AEH25063, AEH25064, AEH25065, AEH25066, AEH25067, AEH25068,AEH25069, AEH25070, AEH25071, AEH25072, AEH25073, AEH25074, AEH25075,and AEH25076. These phytases all have a percentage of identity to anyone of SEQ ID NOs: 2, 4 and 6 of above 70%, however they do not compriseat least one of 119N, 120L, and/or 121E, as defined above.

The sequence of a phytase from Obesumbacterium proteus has beensubmitted to the UNIPROT database with accession number Q6U677. Thisphytase, which is also described by Zinin et al. (2004, FEMSMicrobiology Letters 236: 283-290), has a percentage of identity to SEQID NOs: 2, 4 and 6 of above 70%, however neither does this phytasecomprise at least one of 119N, 120L, and/or 121E, as defined above.

It is an object of the present invention to provide improvedpolypeptides having phytase activity and polynucleotides encoding thepolypeptides. The phytases of the invention have an improved specificactivity, an improved stability such as an improved temperature and/orpH stability, an improved pH activity profile, an improved temperatureactivity profile, an improved substrate profile, an improved performancein animal feed in vitro and/or an improved performance in animal feed invivo.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a polypeptide havingphytase activity and having an amino acid sequence which a) has at least70% identity to amino acids 1-413 of SEQ ID NO: 2, amino acids 1-413 ofSEQ ID NO: 4 and/or amino acids 1-413 of SEQ ID NO: 6 when aligned tothe respective amino acid sequence using the Needle program with theBLOSUM62 substitution matrix, a gap opening penalty of 10.0, and a gapextension penalty of 0.5; and b) comprises at least one of the followingamino acids at the position indicated, 119N, 120L, and/or 121E, whenaligned as described in a) to amino acids 1-413 of SEQ ID NO: 2 andusing an amino acid residue numbering corresponding to amino acids 1-413of SEQ ID NO: 2.

In a particular embodiment, the polypeptide comprises at least one ofthe following amino acids at the position indicated, 109Q, 111G, 119N,120L, and/or 121E.

In another particular embodiment, the polypeptide a) has at least 78%identity, such as, at least 80% identity, at least 85% identity, atleast 90% identity, at least 95% identity, at least 96% identity, atleast 97% identity, at least 98% identity, at least 99% identity toamino acids 1-413 of SEQ ID NO: 2, amino acids 1-413 of SEQ ID NO: 4,and/or amino acids 1-413 of SEQ ID NO: 6; and b) comprises at least oneof the following amino acids at the position indicated: 109Q, 111G,119N, 120L, 121E, and/or 193Q.

In another aspect, the invention relates to a polypeptide having phytaseactivity and having an amino acid sequence which a) has at least 78%identity, such as, at least 80% identity, at least 85% identity, atleast 90% identity, at least 95% identity, at least 96% identity, atleast 97% identity, at least 98% identity, at least 99% identity toamino acids 1-413 of SEQ ID NO: 2, amino acids 1-413 of SEQ ID NO: 4,and/or amino acids 1-413 of SEQ ID NO: 6; and b) comprises at least oneof the following amino acids at the position indicated: 1S, 10I, 38S,66E 71K, 81A, 109Q, 111G, 119N, 120L, 121E, 141R, 142L, 152M, 155E,193Q, 214V, 239K, 245D, 248E, 255A,T, 268A,T, 277T, 283D,E, 285K, 287D,288A,V, 293G, 296S, 303L, 314A, 337I, 345A, 350I, 364A, 371K, 372E,396P, 399K, 406E, and/or 413P.

In another aspect, the invention relates to a polypeptide having phytaseactivity, selected from the group consisting of, (i) a polypeptidecomprising a mature part of SEQ ID NO: 2 (such as the sequence of aminoacids 1-413 of SEQ ID NO: 2); (ii) a variant of (i) comprising at leastone of the following substitutions; K26E, N37Y, A89T, D92E, T134I,V,H160R, S164F, T171I, T176K, A178P, S188N, D190E, A192G, K207E,T, A209S,D211C, A235V, E248L, Q256H,Y, A261 E, N270K, D283N, A288E, I303F, and/orN318D; and (iii) a variant of (i) or (ii) comprising at least one of thefollowing substitutions: N1S, V9I, T38S, E66Q, Q71K, T81A, R141Q, L142V:T152M, E155D, V214I, K239N, D245E, E248S, A255T, R268A,T, A277T, D283E,N285K, T287D, A288V, D293G, P296S, I303L, S314A, I337V, A345S, V350I,A364S, K371N, E372Q, P396S, K399T, E406V, and/or Q413P.

In another aspect, the invention relates to a polypeptide having phytaseactivity, selected from the group consisting of: (i) a polypeptidecomprising a mature part of SEQ ID NO: 4 (such as the sequence of aminoacids 1-413 of SEQ ID NO: 4); (ii) a variant of (i) comprising at leastone of the following substitutions: K26E, N37Y, A89T, D92E, T134I,V,H160R, S164F, T171I. T176K, A178P, S188N, D190E, A192G, K207E,T, A209S,D211C, A235V, S248L, Q256H,Y, A261E, N270K, E283N, V288E, I303F, and/orN318D; and (iii) a variant of (i) or (ii) comprising at least one of thefollowing substitutions: S1N, I9V, S38T, Q66E, K71Q, T81A, Q141 R,V142L, M152T, E155D, I214V, N239K, E245D, S248E, T255A, A268R,T, T277A,E283D, K285N, D287T, V288A, D293G, S296P, I305L, A314S, V337I, S345A,I350V, A364S, N371K, E372Q, S396P, T399K, V406E, and/or P413Q.

Yet another aspect of the invention relates to a polypeptide havingphytase activity, selected from the group consisting of: (i) apolypeptide comprising a mature part of SEQ ID NO: 6 (such as thesequence of amino acids 1-413 of SEQ ID NO: 6); (ii) a variant of (i)comprising at least one of the following substitutions: K26E, N37Y,A89T, D92E, T134I,V, H160R, S164F, T171I, T176K, A178P, S188N, D190E,A192G, K207E,T, A209S, D211C, A235V, S248L, Q256H,Y, A261E, N270K,E283N, V288E, L303F, and/or N 318D; and (iii) a variant of (i) or (ii)comprising at least one of the following substitutions: N1S, V9I, T38S,E66Q, Q71K, A81T, Q141R, V142L, T152M, D155E, I214V, N239K, E245D,S248E, A255T, T268A,R, T277A, E283D, K285N, D287T, V288A, G293D, P296S,L303I, A314S, V337I, S345A, V350I, S364A, N371K, Q372E, S396P, T399K,V406E, and/or Q413P.

Another aspect of the invention is directed to a phytase which has anamino acid sequence which has at least 80% identity, such as, at least85% identity, at least 90% identity, at least 95% identity, at least 96%identity, at least 97% identity, at least 98% identity, at least 99%identity, to amino acids 1-413 of SEQ ID NO: 2, amino acids 1-413 of SEQID NO: 4, or amino acids 1-413 of SEQ ID NO: 6.

In another aspect, the present invention is directed to a phytasevariant which has an amino acid sequence which has at least 70%identity, at least 75% identity, at least 80% identity, such as, atleast 85% identity, at least 90% identity, at least 95% identity, atleast 96% identity, at least 97% identity, at least 98% identity, atleast 99% identity to a polypeptide comprising a mature part (such asthe sequence of amino acids 34-446) of any one of the following GENESEQPsequences: AEH25057, AEH25059, AEH25058, AEH25067, AEH25071, AEH25072,AEH25074, AEH25076, AEH25073, AEH25070, AEH25069, AEH25066, AEH25060,AEH25068, AEH25063, AEH25065, AEH25062, AEH25061, AEH25056, AEH25051, orAEH25064; which variant has phytase activity and comprises at least oneof the following substitutions, N1S, V101, T38S, Q66E, Q71K, T81A,E109Q, H111G, D119N, I120L, K121E, Q141R, V142L, T152M, D155E, L193Q,I214V, N239K, E245D, S248E, V255A,T, R268A,T, A277T, N283D,E, N285K,T287D, E288A,V, D293G, P296S, I303L, A314S, V337I, S345A, V350I, S364A,N371 K, Q372E, S396P, T399K, V406E, and/or Q413P.

In a particular embodiment, the invention relates to a variant of apolypeptide comprising a mature part (such as the sequence of aminoacids 34-446) of GENESEQP:AEH25075, which variant has phytase activityand comprises at least one of the following substitutions: N1S, V10I,T38S, Q66E, Q71K, T81A, E109Q, H111G, D119N, I120L, K121E, Q141R, V142L,T152M, D155E, L193Q, I214V, N239K, E245D, S245E, V255A,T, R268A,T,A277T, N283D,E, N285K, T287D, E288A,V, D293G, P296S, F303L, A314S,V337I, S345A, V350I, S364A, N371K, Q372E, S396P, T399K, V406E, and/orQ413P.

In each of these aspects, for calculating identity and determining aminoacid residue positions, an alignment is produced as described under thefirst aspect above.

The invention also relates to polynucleotides encoding thesepolypeptides, as well as nucleic acid constructs, recombinant expressionvectors, and recombinant host cells comprising the polynucleotides, andto methods for producing and using these polypeptides.

Another aspect of the present invention relates to animal feedcompositions comprising a phytase of the present invention. The presentinvention also provides methods for improving the nutritional value ofan animal feed using a phytase of the present invention.

Another aspect of the present invention relates to methods for treatingproteins, including vegetable proteins, with a phytase of the presentinvention.

Yet another aspect of the present invention relates to the methods forproducing a fermentation product, such as, e.g., ethanol, beer, wine,wherein the fermentation is carried out in the presence of a phytase ofthe present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a multiple alignment of the expected mature parts of SEQ IDNO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 of the invention together with thesequences with the following GENESEQP accession numbers: AEH25051,AEH25056, AEH25057, AEH25058, AEH25059, AEH25060, AEH25061, AEH25062,AEH25063, AEH25064, AEH25065, AEH25066, AEH25067, AEH25068, AEH25069,AEH25070, AEH25071, AEH25072, AEH25073, AEH25074, AEH25075, andAEH25076. The alignment was made using the Clustal method (Higgins,1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software(DNASTAR, Inc., Madison, Wis.) with an identity table and the followingmultiple alignment parameters: Gap penalty of 10 and gap length penaltyof 10. Pairwise alignment parameters are Ktuple=1, gap penalty=3,windows=5, and diagonals=5.

FIG. 2 shows an alignment of UNIPROT accession no, Q6U677 with aminoacids 1-413 of SEQ ID NO: 2. The alignment was made using the programneedle with the matrix BLOSUM62, a gap initiation penalty of 10.0 and agap extension penalty of 0.5.

DETAILED DESCRIPTION OF THE INVENTION Structural Considerations

The structures of three phytases of the invention (mature part of SEQ IDNOs: 2, 4 and 6) were built by homology modelling, using as a templatethe structure of the E. coli AppA phytase (Protein Data Bank id.: 1DKN;Lim et al., 2000, Nat. Struct. Biol. 2: 108-113).

Positions 119 to 121 are facing the active site cleft and therefore thespecific amino acids in these positions are expected to have an effecton specific activity. For increasing the specific activity, 119N isestimated as the better choice, followed by 121E, and finally 120L.

Positions 109 and 111 are close to a disulfide in a region which is abit stressed, so the specific amino acids in these positions areexpected to have an effect on stability, on thermostability inparticular. 111G may be better than 109Q.

Position 193 is another region which may influence the stability of theenzyme. 193Q may provide a good stability.

Corresponding phytase variants, and other phytase variants (e.g., avariant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of amino acids), may be prepared bymethods known in the art and tested as described in the experimentalpart.

Phytase Polypeptides, Percentage of Identity

In the present context a phytase is a polypeptide having phytaseactivity, i.e., an enzyme which catalyzes the hydrolysis of phytate(myo-inositol hexakisphosphate) to (1) myo-inositol and/or (2) mono-,di-, tri-, tetra- and/or penta-phosphates thereof and (3) inorganicphosphate.

In the present context the term a phytase substrate encompasses, i.a.,phytic acid and any phytate (salt of phytic acid), as well as thephosphates listed under (2) above.

The ENZYME site at the internet (www.expasy.ch/enzyme/) is a repositoryof information relative to the nomenclature of enzymes. It is primarilybased on the recommendations of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (IUB-MB) andit describes each type of characterized enzyme for which an EC EnzymeCommission) number has been provided (Bairoch, The ENZYME database,2000, Nucleic Acids Res 28: 304-305). See also the handbook EnzymeNomenclature from NC-IUBMB, 1992).

According to the ENZYME site, three different types of phytases areknown: A so-called 3-phytase (alternative name 1-phytase; a myo-inositolhexaphosphate 3-phosphohydrolase, EC 3.1.3.8), a so-called 4-phytase(alternative name 6-phytase, name based on 1L-numbering system and not1D-numbering, EC 3.1.3.26), and a so-called 5-phytase (EC 3.1.3.72). Forthe purposes of the present invention, all three types are included inthe definition of phytase.

In a particular embodiment, the phytases of the invention belong to thefamily of acid histidine phosphatases, which includes the Escherichiacoli pH 2.5 acid phosphatase (gene appA) as well as fungal phytases suchas Aspergillus awamorii phytases A and B (EC: 3.1.3.8) (gene phyA andphyB). The histidine acid phosphatases share two regions of sequencesimilarity, each centered around a conserved histidine residue. Thesetwo histidines seem to be involved in the enzymes' catalytic mechanism.The first histidine is located in the N-terminal section and forms aphosphor-histidine intermediate while the second is located in theC-terminal section and possibly acts as proton donor.

In a further particular embodiment, the phytases of the invention have aconserved active site motif viz., R-H-G-V-R-A-P (see amino acids 18 to24 of SEQ ID NOs: 2, 4, and 6).

For the purposes of the present invention the phytase activity isdetermined in the unit of FYI, one FYT being the amount of enzyme thatliberates 1 micro-mol inorganic ortho-phosphate per min. under thefollowing conditions: pH 5.5; temperature 37° C.; substrate: sodiumphytate (C₆H₆O₂₄P₆Na₁₂) in a concentration of 10.8 mmol/I. Phytaseactivity is preferably determined using the assay of Example 3 herein.Other suitabte phytase assays are the FYT and FTU assays described inExample 1 of WO 00/20569. FTU is for determining phytase activity infeed and premix.

In a particular embodiment the phytase of the invention is isolated. Theterm “isolated” as used herein refers to a polypeptide which is at least20% pure, preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, most preferably at least90% pure, and even most preferably at least 95% pure, such as, at least96%, 97%, 98%, 99% and higher purity, as determined by SDS-PAGE. Inparticular, it is preferred that the polypeptides are in “essentiallypure form”, i.e., that the polypeptide preparation is essentially freeof other polypeptide material with which it is natively associated. Thiscan be accomplished, for example, by preparing the polypeptide by meansof well-known recombinant methods and/or by classical purificationmethods.

When used herein the term “mature part” refers to that part of thepolypeptide which is secreted by a cell which contains, as part of itsgenetic equipment, a polynucleotide encoding the polypeptide. Generally,the mature polypeptide part refers to that part of the polypeptide whichremains after the N-terminal signal peptide part is cleaved off once ithas fulfilled its function of directing the encoded polypeptide into thecell's secretory pathway. However, experience shows that sometimes alsominor C-terminal truncations occur during the secretion process. Theterm mature part as used herein also takes into account such C-terminaltruncations, if any. The expected signal peptide parts of SEQ ID NOs: 2,4 and 6 are amino acids −33 to −1 of SEQ ID NO: 2, −9 to −1 of SEQ IDNO: 4 (this is a partial signal peptide), and amino acids −33 to −1 ofSEQ ID NO: 6 (see the sequence listing included herewith). SEQ ID NO: 8,which is encoded by SEQ ID NO: 7, is also a signal peptide. As we arenot presently aware of any C-terminal truncations having occurred duringsecretion, the expected mature parts of SEQ ID NOs: 2, 4, and 6 areamino acids 1-413 thereof.

In various aspects of the present invention, the relatedness between twoamino acid sequences is described by the parameter “identity”. Forpurposes of the present invention, the alignment of two amino acidsequences is determined by using the Needle program from the EMBOSSpackage (http://emboss.org), preferably in version 2.8.0. The Needleprogram implements the global alignment algorithm described in Needlemanand Wunsch, 1970, J. Mol. Biol. 48; 443-453. The substitution matrixused is BLOSUM62, the gap opening (initiation) penalty is 10.0, and thegap extension penalty is 0.5.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”) and an amino acid sequence referred toin the claims (e.g., amino acids 1-413 of SEQ ID NO: 2) is calculated asthe number of exact matches in an alignment of the two sequences,divided by the length of the “invention sequence,” or the length ofamino acids 1-413 of SEQ ID NO: 2, whichever is the shortest. The resultis expressed in percent identity.

An exact match occurs when the “invention sequence” and SEQ ID NO: 2have identical amino acid residues in the same positions of the overlap(in the alignment example below this is represented by “|”). The lengthof a sequence is the number of amino acid residues in the sequence(e.g., the length of amino acids 1-413 of SEQ ID NO: 2 is 413).

In the purely hypothetical alignment example below, the overlap is theamino acid sequence “HTWGER-NL” of Sequence 1, or the amino acidsequence “HGWGEDANL” of Sequence 2. In the example a gap is indicated bya “˜”.

Hypothetical Alignment Example:

In a particular embodiment, the percentage of identity of an amino acidsequence of a polypeptide with, or to, e.g., amino acids 1-413 of SEQ IDNO: 2 is determined by i) aligning the two amino acid sequences usingthe Needle program, with the BLOSUM62 substitution matrix, a gap openingpenalty of 10.0, and a gap extension penalty of 0.5; ii) counting thenumber of exact matches in the alignment; iii) dividing the number ofexact matches by the length of the shortest of the two amino acidsequences, and iv) converting the result of the division of iii) intopercentage.

In the above hypothetical example, the number of exact matches is 6, thelength of the shortest one of the two amino acid sequences is 12;accordingly the percentage of identity is 50%.

Another example of an alignment is shown in FIG. 2, where the phytasefrom Obesumbacterium proteus (UNIPROT; Q6U677) is aligned with aminoacids 1-413 of SEQ ID NO: 2. From this alignment it appears that thepercentage of identity of UNIPROT:Q6U677 to amino acids 1-413 of SEQ IDNO: 2 is 76.3% (315 exact matches, the length of the shortest sequenceis 413).

In particular embodiments of the phytase of the invention (i.e.,according to any one of the various aspects thereof), the degree ofidentity to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6 is at least71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orat least 99%. In still further particular embodiments, the degree ofidentity is at least 90.0%, 90.2%, 90.4%, 90.6%, 90.8%, 91.0%, 91.2%,91.4%, 91.6%, 91.8%, 92.0%, 92.2%, 92.4%, 92.6%, 92.8%, 93.0%, 93.2%,93.4%, 93.6%, 93.8%, 94.0%, 94.2%, 94.4%, 94.6%, 94.8%, 95.0%, 95.2%,95.4%, 95.6%, 95.8%, 96.0%, 96.2%, 96.4%, 96.6%, 96.8%, 97.0%, 97.2%,97.4%, 97.6%, 97.8%, 98.0%, 98.2%, 98.4%, 98.6%, 98.8%, 99.0%, 99.2%,99.4%, 99.6%, or at least 99.8%.

In still further particular embodiments, the phytase of the inventionhas (or has an amino acid sequence which differs by) no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or no more than 10 alterations as compared to anyone of amino acids 1-413 of SEQ ID NO: 2, amino acids 1-413 of SEQ IDNO: 4, and amino acids 1-413 of SEQ ID NO: 6; no more than 11, 12, 13,14, 15, 16, 17, 18, 19, or no more than 20 alterations as compared toany one of amino acids 1-413 of SEQ ID NO: 2, amino acids 1-413 of SEQID NO: 4, and amino acids 1-413 of SEQ ID NO: 6; no more than 21, 22,23, 24, 25, 26, 27, 28, 29, or no more than 30 alterations as comparedto any one of amino acids 1-413 of SEQ ID NO: 2, amino acids 1-413 ofSEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6: no more than 31,32, 33, 34, 35, 36, 37, 38, 39, or no more than 40 alterations ascompared to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6; no morethan 41, 42, 43, 44, 45, 46, 47, 48, 49, or no more than 50 alterationsas compared to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6, no morethan 51, 52, 53, 54, 55, 56, 57, 58, 59, or no more than 60 alterationsas compared to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6; no morethan 61, 62, 63, 64, 65, 66, 67, 68, 69, or no more than 70 alterationsas compared to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6; no morethan 71, 72, 73, 74, 75, 76, 77, 78, 79, or no more than 80 alterationsas compared to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6; no morethan 81, 82, 83, 84, 85, 86, 87, 88, 89, or no more than 90 alterationsas compared to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6, no morethan 91, 92, 93, 94, 95, 96, 97, 98, 99, or no more than 100 alterationsas compared to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6; no morethan 101, 102, 103, 104, 105, 106, 107, 108, 109, or no more than 110alterations as compared to any one of amino acids 1-413 of SEQ ID NO: 2,amino acids 1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO:6; no more than 111, 112, 113, 114, 115, 116, 117, 118, 119, or no morethan 120 alterations as compared to any one of amino acids 1-413 of SEQID NO: 2, amino acids 1-413 of SEQ ID NO: 4, and amino acids 1-413 ofSEQ ID NO: 6; or no more than 121, 122, 123, or 124 alterations ascompared to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6.

In alternative embodiments of the phytase of the invention, the degreeof identity to any one of amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6 is at least55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, orat least 69%.

Position Numbering

The nomenclature used herein (i.e., according to any one of the variousaspects of the invention) for defining amino acid positions is based onthe amino acid sequence of the phytase derived from Buttiauxellagaviniae DSM 18930, viz., the expected mature sequence thereof which isamino acids 1-413 of SEQ ID NO: 2. Accordingly, in the present context,the basis for numbering positions is SEQ ID NO: 2 starting with N1 andending with Q413. This position numbering is shown as the first line inthe alignment of FIG. 1 and this numbering is also shown in thealignment of FIG. 2 (the upper row of the alignment).

Alterations, such as Substitutions, Deletions, Insertions

A phytase of the invention (i.e., according to any one of the variousaspects of the invention), be it a wildtype or a variant, can comprisevarious types of alterations relative to a template (i.e., a referenceor comparative amino acid sequence such as amino acids 1-413 of SEQ IDNO: 2). An amino acid can be substituted with another amino acid; anamino acid can be deleted; an amino acid can be inserted; as well as anycombination of any number of such alterations. In the present contextthe term “insertion” is intended to cover also N- and/or C-terminalextensions, and the term “deletion” is intended to cover also N- and/orC-terminal truncations.

The general nomenclature used herein for a single alteration is thefollowing: XDcY, where “X” and “Y” independently designate a one-letteramino acid code, or a “*”, “D” designates a number, and “c” designatesan alphabetical counter (a, b, c, and so forth), which is only presentin insertions. Reference is made to Table 1 below which describes purelyhypothetical examples of applying this nomenclature to various types ofalterations.

TABLE 1 Type Description Example Subsutution X = Amino acid in templateD= Position in templatec emptyY = Amino acid in variant

Insertion X = “*”D = Position in templatebefore the insertionc = “a” forfirst insertion atthis position, “b” for next,etc

Deletion X = Amino acid in templateD = Position in template c emptyY =“*”

N-terminalextension Insertions at position “0”.

C-terminalextension Insertions after the N-terminal amino acid.

As explained above, the position number (“D”) is counted from the firstamino acid residue of amino acids 1-413 of SEQ ID NO: 2.

Several alterations in the same sequence are separated by “/” (slash),e.g. the designation “1*/2*/3*” means that the amino acids in positionnumber 1, 2, and 3 are all deleted, and the designation “104A/105F”means that the amino acid in position number 104 is substituted by A,and the amino acid in position number 105 is substituted by F.

Alternative alterations are separated by “,” (comma), e.g., thedesignation “255A,T” means that the amino acid in position 255 issubstituted with A or T.

The commas used herein in various other enumerations of possibilitiesmean what they usually do grammatically, viz., often and/or. Forexample, the first comma in the listing “255A,T, 406E, and/or 413P”denotes an alternative (A or T), whereas the two next commas should beinterpreted as and/or options: 255A or 255T, and/or 406E, and/or 413P.

In the present context, “at least one” (e.g., amino acids at theposition indicated, or substitution) means one or more, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acids (or substitutions); or 12, 14, 15,16, 18, 20, 22, 24, 25, 28, or 30 amino acids (or substitutions); and soon, up to a maximum number of alterations of 125, 130, 140, 150, 160,170, 180, 190, or of 200.

A substitution or extension without any indication of what to substituteor extend with refers to the insertion of any natural, or non-natural,amino acid, except the one that occupies this position in the template.

Identifying Corresponding Position Numbers

As explained above, the mature phytase of Buttiauxella gaviniae DSM18930 (SEQ ID NO: 2) is used herein as the standard for positionnumbering and, thereby, also for the nomenclature.

For another phytase, be it a wildtype or a variant, the positioncorresponding to position D in SEQ ID NO: 2 is found by aligning the twosequences as specified above in the section entitled “Phytasepolypeptides, percentage of identity”. From the alignment, the positionin the sequence of the invention corresponding to position D of SEQ IDNO: 2 can be clearly and unambiguously identified (the two positions ontop of each other in the alignment).

Here are some purely hypothetical examples derived from Table 1 abovewhich in the third column includes a number of alignments of twosequences.

Consider the third cell in the first row of Table 1: The upper sequenceis the template, the lower the variant. Position number 80 refers toamino acid residue G in the template. Amino acid A occupies thecorresponding position in the variant. Accordingly, this substitution isdesignated G80A.

Consider now the third cell in the second row of Table 1: The uppersequence is again the template and the lower the variant. Positionnumber 80 again refers to amino acid residue G in the template. Thevariant has two insertions, viz., TY, after G80 and before V81 in thetemplate. Whereas the T and Y of course would have their own “real”position number in the variant amino acid sequence, for the presentpurposes we always refer to the template position numbers, andaccordingly the T and the Y are said to be in position number 80a and80b, respectively.

Finally, consider the third cell in the last row of Table 1: Positionnumber 275 refers to the last amino acid of the template. A C-terminalextension of ST are said to be in position number 275a and 275b,respectively, although, again, of course they have their own “real”position number in the variant amino acid sequence.

A real example of such alignment is shown in FIG. 2, where the phytasefrom Obesumbacterium proteus (UNIPROT:Q6U677) is aligned with aminoacids 1-413 of SEQ ID NO: 2. From this alignment it is inferred that,i.a., amino acids 119N, 120L, and 121E of SEQ ID NO: 2 correspond to119D, 120I and 120K of UNIPROT:Q6U677, respectively.

VARIOUS EMBODIMENTS

A polypeptide according to the second aspect of the invention has anamino acid sequence which a) has at least 78% identity to amino acids1-413 of SEQ ID NO: 2, amino acids 1-413 of SEQ ID NO: 4, and/or aminoacids 1-413 of SEQ ID NO: 6; and b) comprises at least one of thefollowing amino acids at the position indicated: 1S, 10I, 38S, 66E, 71K,81A, 109Q, 111G, 119N, 120L, 121E, 141R, 142L, 152M, 155E, 193Q, 214V,239K, 245D, 248E, 255A,T, 268A,T, 277T, 283D,E, 285K, 287D, 288A,V,293G, 296S, 303L, 314A, 337I, 345A, 350I, 364A, 371K, 372E, 396P, 399K,406E, and/or 413P.

Each of the amino acids at the positions indicated under b) above make adifference to the Buttiauxella NCIMB 41248 wildtype phytase and itsvariants with the following GENESEQP accession numbers: AEH25051,AEH25056, AEH25057, AEH25058, AEH25059, AEH25060, AEH25061, AEH25062,AEH25063, AEH25064, AEH25065, AEH25066, AEH25067, AEH25068, AEH25069,AEH25070, AEH25071, AEH25072, AEH25073, AEH25074, AEH25075, andAEH25076.

In particular embodiments thereof, the amino acid sequence comprises a)at least one of 1S, 10I, 38S, 71K: 109Q, 111G, 119N, 120L, 121E, 152M,155E, 193Q, 255T, 268A, 277T, 283E, 285K, 287D, 288V, 296S, 364A, 350I,372E, and/or 413P (as represented by SEQ ID NO: 4); b) at least one of66E, 81A, 109Q, 111G, 119N, 120L, 121E, 193Q, 255A, 268T, 277T, 283E,285K, 287D, 288V, 293G, and/or 303L (as represented by SEQ ID NO: 6);and c) at least one of 66E, 109Q, 111G, 119N, 120L, 121E, 141R, 142L,155E, 193Q, 214V, 239K, 245D, 248E, 255A, 283D, 288A, 314S, 337I, 345A,364A, 371K, 372E, 396P, 399K, and/or 406E (as represented by SEQ ID NO:2).

In another particular embodiment the polypeptide of the invention hasbeen altered inspired by the Buttiauxella wildtype and mutant phytasesdisclosed in WO 2006/043178, viz., it has an amino acid sequence whichcomprises at least one of the following amino acids at the positionindicated; 26E, 37Y, 89T, 92E, 134I,V, 160R, 164F, 171I, 176K, 178P,188N, 190E, 192G, 207E,T, 209S, 211C, 235V, 248L, 256H,Y, 261E, 270K,303F, and/or 318D.

For example, the mature part of SEQ ID NO: 2 is mutated to comprise atleast one of the following substitutions; K26E, N37Y, A89T, D92E,T134I,V, H160R, S164F, T171I, T176K, A178P, S188N, D190E, A192G,K207E,T, A209S, D211C, A235V, E248L, Q256H,Y, A261E, N270K, D283N,A288E, I303F, and/or N318D. Other preferred substitutions in the maturepart of SEQ ID NO: 2 are inspired by SEQ ID NOs: 4 and 6: N1S, V9I,T38S, E66Q, Q71K, T81A, R141Q, L142V, T152M, E155D, V214I, K239N, D245E,E248S, A255T, R268A,T, A277T, D283E, N285K, T287D, A288V, D293G, P296S,I303L, S314A, I337V, A345S, V350I, A364S, K371N, E372Q, P396S, K399T,E406V, and/or Q413P.

As another example, the mature part of SEQ ID NO: 4 is mutated tocomprise at least one of the following substitutions: K26E, N37Y, A89T,D92E, T134I,V, H160R, S164F, T171I, T176K, A178P, S188N, D190E, A192G,K207E,T, A209S, D211C, A235V, S248L, Q256H,Y, A261E, N270K, E283N,V288E, I303F, and/or N318D. Other preferred substitutions in the maturepart of SEQ ID NO: 2 are inspired by SEQ ID NOs: 2 and 6: S1N, I9V,S38T, Q66E, K71Q, T81A, Q141R, V142L, M152T, E155D, I214V, N239K, E245D,S248E, T255A, A268R,T, T277A, E283D, K285N, D287T, V288A, D293G, S296P,I303L, A314S, V337I, S345A, I350V, A364S, N371K, E372Q, S396P, T399K,V406E, and/or P413Q.

As a still further example, the mature part of SEQ ID NO: 6 is mutatedto comprise at least one of the following substitutions: K26E, N37Y,A89T, D92E, T134I,V, H160R, S164F. T171I, T176K, A178P, S188N, D190E,A192G, K207E,T, A209S, D211C, A235V, S248L, Q256H,Y, A261E, N270K,E283N, V288E, L303F, and/or N318D. Other preferred substitutions in themature part of SEQ ID NO: 2 are inspired by SEQ ID NOs: 2 and 4: N1S,V91, T38S, E66Q, Q71K, A81T, Q141R, V142L, T152M, D155E, 1214V, N239K,E245D, S248E, A255T, T268A,R, T277A, E283D, K285N, D287T, V288A, G293D,P296S, L303I, A314S, V337I, S345A, V350I, S364A, N371K, Q372E, S396P,T399K, V406E, and/or Q413P.

As explained above, various aspects of the present invention relate topolypeptides “having” an amino acid sequence which has a specifieddegree of identity to amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and/or amino acids 1-413 of SEQ ID NO: 6, whichhave phytase activity (hereinafter “homologous polypeptides”), or topolypeptides “having” an amino acid sequence which differs by a maximumnumber of amino acids amino acids 1-413 of SEQ ID NO: 2, amino acids1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ ID NO: 6.

A polypeptide of the present invention preferably comprises any one ofamino acids 1-413 of SEQ ID NO: 2, amino acids 1-413 of SEQ ID NO: 4, oramino acids 1-413 of SEQ ID NO: 6; or an allelic variant thereof; or afragment thereof that has phytase activity.

In another preferred embodiment, a polypeptide of the present inventionconsists of the amino acid sequence of amino acids 1-413 of SEQ ID NO:2, amino acids 1-413 of SEQ ID NO: 4, and amino acids 1-413 of SEQ IDNO: 6; or an allelic variant thereof, or a fragment thereof that hasphytase activity.

Anyone of the nucleotide sequences of SEQ ID NO: 1, 3 and 5, or asubsequence thereof, as well as the amino acid sequences of SEQ ID NO:2, 4, and 6, or a fragment thereof, may be used to design a nucleic acidprobe to identify and clone DNA encoding polypeptides having phytaseactivity from strains of different genera or species according tomethods well known in the art. In particular, such probes can be usedfor hybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Both DNA and RNAprobes can be used. The probes are typically labelled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).

A genomic DNA library prepared from such other organisms may, therefore,be screened for DNA which hybridizes with the probes described above andwhich encodes a polypeptide having phytase activity. Genomic or otherDNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO: 1,3 or 5 or a subsequence thereof, the carrier material is used in aSouthern blot.

In a preferred embodiment, the nucleic acid probe comprises nucleotides454-462 of SEQ ID NO: 1, nucleotides 384-392 of SEQ ID NO: 3, ornucleotides 454-462 of SEQ ID NO: 5 (all corresponding to the motif119N, 120L, and 121E). In another preferred aspect, the nucleic acidprobe is a polynucleotide sequence which encodes the polypeptides of anyone of SEQ ID Nos: 2, 4, or 6, or a subsequence thereof. In anotherpreferred aspect, the nucleic acid probe is SEQ ID NO: 1, 3 or 5.

The present invention also relates to variants comprising a conservativesubstitution, deletion, and/or insertion of one or more amino acids inthe sequences of amino acids 1-413 of SEQ ID NO: 2, amino acids 1-413 ofSEQ ID NO: 4, or amino acids 1-413 of SEQ ID NO: 6, or the maturepolypeptides thereof. Preferably, amino acid changes are of a minornature, that is conservative amino acid substitutions or insertions thatdo not significantly affect the folding and/or activity of the protein;small deletions, typically of one to about 30 amino acids; a smallamino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue, a small linker peptide of up to about 20-25residues; or a small extension that facilitates purification by changingnet charge or another function, such as a poly-histidine tract, anantigenic epitope or a binding domain. A fragment is a preferred exampleof a variant comprising a deletion, as described above, and a fragmentpreferably retains phytase activity.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by Neurath and Hill,1979, In, The Proteins, Academic Press, New York. The most commonlyoccurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,phytase activity) to identify amino acid residues that are critical tothe activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labelling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides which are related to apolypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241; 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30: 10832-108371; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireal., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells. Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.In particular embodiments, the polypeptide is obtainable from theProteobacteria; Gammaproteobacteria; Enterobacteriales; family ofEnterobacteriaceae; preferably from the genus Buttiauxella, e.g.,selected from amongst the species of Buttiauxella agrestis, Buttiauxellabrennerae, Buttiauxella ferragutiae, Buttiauxella gaviniae, Buttiauxellaizardii, Buttiauxella noackiae, Buttiauxella warmboldiae, Buttiauxellasp. B22, Buttiauxella sp. BTN01, Buttiauxella sp. LBV 449. Buttiauxellasp. P5, Buttiauxella sp. PNBS, Buttiauxella sp. S212, Buttiauxella sp.S215, Buttiauxella sp. S218. www.ncbi.nlm.nih.gov/Taxonomy/Browser.

In a more preferred aspect, the polypeptide is a Buttiauxella gavniae orButtiauxella agrestis polypeptide, most preferably a Buttiauxellagaviniae DSM 18930, a Buttiauxella agrestis DSM 18931, or a Buttiauxellaagrestis DSM 18932 polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of another microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Altered Properties

In a particular embodiment of any aspect of the invention the phytasehas altered preferably improved, properties. Examples of altered,preferably improved, properties are pI, temperature and/or pH stability,pH activity profile, temperature activity profile, substrate profile,and performance in animal feed in vitro or in vivo.

The terms “altered”, “amended” and “improved” imply a comparison withanother phytase. Examples of such other, reference, or comparative,phytases are the Buttliauxella NCIMB 41248 wildtype phytase and itsvariants with the following GENESEQP accession numbers, AEH25051,AEH25056, AEH25057, AEH25058, AEH25059, AEH25060, AEH25061, AEH25062,AEH25063, AEH25064, AEH25065, AEH25066, AEH25067, AEH25068, AEH25069,AEH25070, AEH25071, AEH25072, AEH25073, ASH 25074, AEH25075, andAEH25076.

The phytases of the invention which comprise at least one of thefollowing amino acids at the position indicated: 119N, 120L, and/or 121Ehave an improved specific activity as shown in the experimental part. Inparticular embodiments, the phytase of the invention comprises i) 119N;ii) 121E; iii) 120L, iv) 119N and 121E; v) 119N and 120L; vi) 121E and120L; or vii) 119N, 120L, and 121E. In still further particularembodiments the phytases of i)-vii) have an improved specific activity.Specific activity is determined as described below.

The phytases of the invention which comprise at least one of thefollowing amino acids at the position indicated: 109Q, and/or 111G areexpected to have an improved stability, in particular an improvedthermostability. In particular embodiments, the phytase of the inventioncomprises i) 111G; ii) 109Q; or iii) 111G and 109Q. In still furtherparticular embodiments the phytases of i)-iii) have an improvedstability, preferably an improved thermostability. Thermostability isdetermined as described below.

The phytases of the invention which comprise 193Q are also expected tohave an improved stability, preferably an improved thermostability.Thermostability is determined as described below.

Additional specific amino acids at specified positions, as well asadditional specific amino acid substitutions are also disclosed herein,which characterize and distinguish additional phytases of the inventionwhich may have improved properties such as pI, temperature stability, pHstability, pH activity profile, temperature activity profile, substrateprofile, and/or improved performance in animal feed in vitro or in vivo.

Specific Activity

In a particular embodiment, the phytase of the invention has an improvedspecific activity relative to a reference phytase. More in particular,the specific activity of a phytase of the invention is at least 105%,relative to the specific activity of a reference phytase determined bythe same procedure. In still further particular embodiments, therelative specific activity is at least 110, 115, 120, 125, 130, 140,145, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350 or even400%, still relative to the specific activity of the reference phytaseas determined by the same procedure.

Examples of reference phytases are listed above. Preferred referencephytases for this embodiment are GENESEQP:AEH25072 (the variantdisclosed in Table 3 of WO 2006/043178), and GENESEQP:AEH25051 (thewildtype phytase from Buttiauxella P1-29 disclosed in Example 10 of WO2006/043178).

In the alternative, the term high specific activity refers to a specificactivity of at least 240 FYT/mg Enzyme Protein (EP). In particularembodiments, the specific activity is at least 300, 350, 400, 450, 500,550, 600, or at least 650 FYT/mg ER.

Specific activity is measured on highly purified samples (an SDS polyacryl amide gel should show the presence of only one component).Preferably, the sample of the enzyme is at least 95% pure, as determinedby SOS-PAGE. The enzyme protein concentration may be determined by aminoacid analysis, and the phytase activity in the units of FYT, determinedas described in Example 3, i.e., on the substrate of sodium phytate atpH 5.5 and 37° C. Specific activity is a characteristic of the specificphytase in question, and it is calculated as the phytase activitymeasured in FYT units per mg phytase variant enzyme protein. See Example4 for further details.

Isoelectric Point (pI)

In particular embodiments, the pI for the phytase of the invention is inthe range of i) 7.0-8.0; ii) 7.2-7.8; or iii) 7.4-7.6. The pI isdetermined as described in Example 5.

pH Profile

In a particular embodiment, the phytase of the invention has an alteredpH profile as compared to a reference phytase. Examples of referencephytases are listed above.

In particular embodiments, the phytase of the invention has a relativeactivity at pH 2.0 which is at least 30% of the activity at pH 4.5(optimum pH), preferably at least 31%, 32%, 33%, 34% 35%, 36%, 37%, 38%,39%, 40%, or at least 41%.

In additional particular embodiments, the phytase of the invention has arelative activity at pH 6.0 which is at least 10% of the activity at pH4.5 (optimum pH), preferably at least 15%, 20%, 25%, 30%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, or at least 43%.

The pH profile (phytase activity as a function of pH) is determined onhighly purified samples (an SDS poly acryl amide get should show thepresence of only one component). Preferably, the sample of the enzyme isat least 95% pure, as determined by SDS-PAGE. The phytase activity isdetermined in the pH range of 2.0 to 7.5 using a buffer cocktail (50 mMglycine, 50 mM acetic acid and 50 mM Bis-Tris (Bis-(2-hydroxyethyl)imino-tris (hydroxymethyl) methan), and on the substrate of sodiumphytate at pH 5.5 and 37° C. A preferred assay is the assay of Example3, except that the buffer is replaced with the above indicated buffer.More details are found in Example 6

pH Stability

In a particular embodiment, the phytase of the invention has an alteredpH stability as compared to a reference phytase. Examples of referencephytases are listed above.

In particular embodiments, the phytase of the invention has a residualphytase activity after incubation for 1½ hours at 40° C. and at pHselected from 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 of at least 50%,preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 90%,92%, or at least 94%, relative to the phytase activity at 0 hours(before start of the incubation). In preferred embodiments, theincubation pH is i) 2.0, ii) 3.0, iii) 4.0, iv) 5.0, v) 6.0, vi) 7.0,and vii) 8.0. In even more preferred embodiments, the incubation pH is2.0, and the residual activity is at least 50%, 55%, 60%, 65%, 70%, 75%,80%, or at least 82%. The phytase is incubated in 0.1 M glycine, 0.1 Macetic acid, 0.1 M Bis-Tris, adjusted to the desired pH. The phytaseactivity is determined on the substrate of sodium phytate at pH 5.5 and37° C. The activity assay of Example 3 where the buffer is replaced withthe above indicated buffer is a preferred assay. More details are foundin Example 7.

In still further particular embodiments, the phytase of the inventionhas a residual phytase activity after incubation for 24 hours at 40° C.and at pH selected from 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 of atleast 50%, preferably at least 51%, 52%, 54%, 56%, 58%, 60%, 62%, 64%,66%, 68%, 70%, 72%, 73% 74%, 75%, 76%, 77%, 78%, 79%, 80%, 82%, 84%,86%, or at least 88%, relative to the phytase activity at 0 hours(before start of the incubation). In preferred embodiments, theincubation pH is i) 2.0, ii) 3.0, iii) 4.0, iv) 5.0, v) 6.0, vi) 7.0,and vii) 8.0. In even more preferred embodiments, the incubation pH is2.0, and the residual activity is at least 50%, 51%, 52%, 54%, or atleast 56%. The phytase is incubated in 0.1 M glycine, 0.1 M acetic acid,0.1 M Bis-Tris, adjusted to the desired pH. The phytase activity isdetermined on the substrate of sodium phytate at pH 5.5 and 37° C. Theactivity assay of Example 3 where the buffer is replaced with the aboveindicated buffer is a preferred assay. More details are found in Example7.

Temperature Profile

In a particular embodiment, the phytase of the invention has an alteredtemperature profile as compared to a reference phytase. Examples ofreference phytases are listed above.

In particular embodiments, the phytase of the invention has a relativeactivity at 30° C. which is at least 20% of the activity at 60° C.(optimum temperature), preferably at least 22%, 24%, 26%, 28%, 30%, 32%,34%, 36%, 38%, or at least 40%.

In additional particular embodiments, the phytase of the invention has arelative activity at 70° C. which is at least 10% of the activity at 60°C. (optimum temperature), preferably at least 12%, 14%, 16%, 18%, 20%,22%, or at least 23%.

The temperature profile (phytase activity as a function of temperaturesis determined on highly purified samples (an SDS poly acryl amide gelshould show the presence of only one component). Preferably, the sampleof the enzyme is at least 95% pure, as determined by SOS-PAGE. Thephytase activity is determined in the temperature range of 20-90° C. atpH 4.0, in the alternative at pH 5.5. In both cases a 0.25 M sodiumacetate buffer is used. The activity is determined on the substrate ofsodium phytate. A preferred assay is the assay of Example 3, except ofcourse that the temperature differs, and if desired also the pH, cf.above. More details are found in Example 8.

Thermostability

In a particular embodiment, the phytase of the invention has an altered,preferably improved, thermostability as compared to a reference phytase.Examples of reference phytases are listed above.

In particular embodiments, the phytase of the invention has a residualphytase activity after incubation for a desired time (such as 15minutes, 30 minutes, 1 hour, 1½ hours, or 2 hours) at an elevatedtemperature (such as 65, 70, 75, 80, 85, 90, or 95° C.) at pH 4.5 of atleast 50%, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%,86%, 90%, 92%, or at least 94%, relative to the phytase activity at 0hours (before start of the incubation). In a preferred embodiment, theincubation time is 1 hour, the pH is 4.5 and the temperature 80° C. Thephytase is incubated in 0.25 M sodium acetate. The phytase activity isdetermined on the substrate of sodium phytate at pH 5.5 and 37° C. Theactivity assay of Example 3 is a preferred assay.

In the alternative, Differential Scanning Calorimetry (DSC) measurementsmay be used to determine the denaturation temperature, Td, of thepurified phytase protein. The Td is indicative of the thermostability ofthe protein: The higher the Td, the higher the thermostability. DSCmeasurements may be performed at various pH values, e.g., using theVP-DSC from Micro Cal. Scans are performed at a constant scan rate of1.5 C/min from 20-90° C. Preferred pH values are 4.0 and 5.5, preferably4.0. Before running the DSC, the phytases are desalted, e.g., usingNAP-5 columns (Pharmacia) equilibrated in appropriate buffers (e.g., 25mM sodium acetate pH 4.0; 0.1 M sodium acetate, pH 5.5). Data-handlingis performed using the MicroCal Origin software (version 4.10), and thedenaturation temperature, Td (also called the melting temperature, Tm)is defined as the temperature at the apex of the peak in the thermogram.

In particular embodiments, the phytase of the invention has a Td, whichmay be determined as described above, of at least 60° C. In stillfurther particular embodiments, the Td is at least 61, 62, 64, 66, 68,70, 72, 74, 76, 78, or at least 80° C.

Performance in Animal Feed

In a particular embodiment the phytase of the invention has an improvedperformance in animal feed as compared to a reference phytase. Examplesof reference phytases are listed above.

The performance in animal feed may be determined using an in vitro modelsimulating the gastro-intestinal conditions in a monogastric animal,e.g., as follows:

Feed samples composed of 30% soybean meal and 70% maize meal with addedCaCl₂ to a concentration of 5 g calcium per kg feed are prepared andpreincubated at 40° C. and pH 3.0 for 30 minutes followed by addition ofpepsin (3000 U/g feed) and suitable dosages of the phytases (identicaldosages are used for all phytases to be tested to allow comparison), forexample between 0.25 to 0.75 phytase units FYT/g feed. A blank with nophytase activity is also included as reference. The samples are thenincubated at 40° C. and pH 3.0 for 60 minutes followed by pH 4.0 for 30minutes.

The reactions are stopped and phytic acid and inositol-phosphatesextracted by addition of HCl to a final concentration of 0.5 M andincubation at 40° C. for 2 hours, followed by one freeze-thaw cycle and1 hour incubation at 40° C.

Phytic acid and inositol-phosphates are separated by high performanceion chromatography as described by Chen et al. (2003, Journal ofChromatography A 1018: 41-52) and quantified as described by Skoglund etal. (1997, J. Agric. Food Chem. 45: 431-436).

Released phosphorous is then calculated as the difference ininositol-phosphate bound phosphorous (IP-P) between phytase-treated andnon-treated samples. The relative performance of a specified phytase iscalculated as the percentage of the phosphorous released relative to thedesired reference phytase.

In particular embodiments the relative performance in vitro of thephytase of the invention is at least 105%, preferably at least 110, 120,130, 140, 150, 160, 170, 180, 190, or at least 200%.

Polynucleotides, Nucleic Acid Constructs, and Expression Vectors

The present invention also relates to polynucleotides comprising anucleotide sequence which encode a polypeptide of the present invention.The polynucleotides are preferably substantially pure, or isolated,which refers to a polynucleotide preparation free of other extraneous orunwanted nucleotides and in a form suitable for use within geneticallyengineered protein production systems. Thus, a substantially purepolynucleotide contains at most 10%, preferably at most 8%, morepreferably at most 6%, more preferably at most 5%, more preferably atmost 4%, more preferably at most 3%, even more preferably at most 2%,most preferably at most 1%, and even most preferably at most 0.5% byweight of other polynucleotide material with which it is nativelyassociated. A substantially pure polynucleotide may, however, includenaturally occurring 5′ and 3′ untranslated regions, such as promotersand terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

The term “nucleic acid construct” as used herein refers to a nucleicacid molecule, either single or doubt-stranded, which is isolated from anaturally occurring gene or which is modified to contain segments ofnucleic acids in a manner that would not otherwise exist in nature. Theterm nucleic acid construct is synonymous with the term “expressioncassette” when the nucleic acid construct contains the control sequencesrequired for expression of a coding sequence of the present invention.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide encoding a polypeptide of the present invention. Eachcontrol sequence may be native or foreign to the nucleotide sequenceencoding the polypeptide. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican 1980, 242: 74-94, and in Sambrook et al., 1989, supra.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva 1993. Microbiological Reviews 57:109-137.

In a preferred aspect, the signal peptide coding region is nucleotides 1to 81 of SEQ ID NO: 7 which encode amino acids 1 to 27 of SEQ ID NO: 8.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. Other examples of regulatory sequencesare those which allow for gene amplification. In these cases, thenucleotide sequence encoding the polypeptide would be operably linkedwith the regulatory sequence.

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The term “expressionvector” is defined herein as a linear or circular DNA molecule thatcomprises a polynucleotide encoding a polypeptide of the invention, andwhich is operably linked to additional nucleotides that provide for itsexpression. The various nucleic acids and control sequences describedabove may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleotide sequence encoding thepolypeptide at such sites. Alternatively, a nucleotide sequence of thepresent invention may be expressed by inserting the nucleotide sequenceor a nucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector wilt typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

A vector comprising a polynucleotide of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.

The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

A conditionally essential gene may function as a non-antibioticselectable marker. Non-limiting examples of bacterial conditionallyessential non-antibiotic selectable markers are the dal genes fromBacillus subtilis, Bacillus licheniformis, or other Bacilli, that areonly essential when the bacterium is cultivated in the absence ofD-alanine. Also the genes encoding enzymes involved in the turnover ofUDP-galactose can function as conditionally essential markers in a cellwhen the cell is grown in the presence of galactose or grown in a mediumwhich gives rise to the presence of galactose. Non-limiting examples ofsuch genes are those from B. subtilis or B. licheniformis encodingUTP-dependent phosphorylase (EC 2.7.7.10), UDP-glucose-dependenturidylyltransferase (EC 2.7.7.12) or UDP-galactose epimerase (EC5.1.3.2).

Also a xylose isomerase gene such as xylA, of Bacilli can be used asselectable markers in cells grown in minimal medium with xylose as solecarbon source. The genes necessary for utilizing gluconate, gntK, andgntP can also be used as selectable markers in cells grown in minimalmedium with gluconate as sole carbon source. Other examples ofconditionally essential genes are known in the art. Antibioticselectable markers confer antibiotic resistance to such antibiotics asampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline,neomycin, hygromycin or methotrexate.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replications” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cents, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. The term “host cell”,as used herein, includes any cell type which is susceptible totransformation, transfection, transduction, and the like with a nucleicacid construct comprising a polynucleotide of the present invention.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a cell,which in its wild-type form is capable of producing the polypeptide,under conditions conducive for production of the polypeptide, and (b)recovering the polypeptide. Preferably, the cell is of the genusButtiauxella, and more preferably Buttiauxella agrestis or Buttiauxellagaviniae.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, Janson and Ryden, editors, VCH Publishers, NewYork, 1989). A typical purification scheme may include centrifugation,germ filtration, ammonium sulphate precipitation (using: e.g., 80%ammonium sulphate saturation), centrifugation, re-suspension of pelletsin 50 mM sodium acetate buffer pH 4.5, filtration, dialysis against 50mM sodium acetate buffer and cation exchange chromatography(S-sepharose: loading with 50 mM sodium acetate pH 4.5, eluting with alinear salt gradient (50 mM sodium acetate pH 4.5+1 M NaCl)).

Transgenic Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having phytase activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

In a particular embodiment, the polypeptide is targeted to the endospermstorage vacuoles in seeds. This can be obtained by synthesizing it as aprecursor with a suitable signal peptide, see Horvath et al. (2000, PNAS97(4): 1914-1919.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot) or engineered variants thereof. Examples of monocot plantsare grasses, such as meadow grass (blue grass, Poa), forage grass suchas Festuca, Lolium, temperate grass, such as Agrostis, and cereals,e.g., wheat, oats, rye, barley, rice, sorghum, triticale (stabilizedhybrid of wheat (Triticum) and rye (Secale), and maize (corn). Examplesof dicot plants are tobacco, legumes, such as sunflower (Helianthus),cotton (Gossypium), lupins, potato, sugar beet, pea, bean and soybean,and cruciferous plants (family Brassicaceae), such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana.Low-phytate plants as described, e.g., in U.S. Pat. Nos. 5,689,054 and6,111,168 are examples of engineered plants.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers, as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyma vascular tissues, meristems. Alsospecific plant cell compartments, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part. Likewise, plant parts such as specifictissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleic acid sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleic acid sequence in the plant or plant partof choice. Furthermore, the expression construct may comprise aselectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences are determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific cell compartment, tissue or plant partsuch as seeds or leaves. Regulatory sequences are, for example,described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the following promoters may be used: The35S-CaMV promoter (Franck et al., 1980, Cell 21; 285-294), the maizeubiquitin 1 (Christensen, Sharrock and Quail, 1992. Maize polyubiquitingenes: structure, thermal perturbation of expression and transcriptsplicing, and promoter activity following transfer to protoplasts byelectroporation), or the rice actin 1 promoter (Plant Mol. Biol. 18,675-689.; Zhang, McElroy and Wu, 1991, Analysis of rice Act1 5′ regionactivity in transgenic rice plants, Plant Cell 3: 1155-1185).Organ-specific promoters may be, for example, a promoter from storagesink tissues such as seeds, potato tubers, and fruits (Edwards andCoruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sinktissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24:863-878), a seed specific promoter such as the glutelin, prolamin,globulin, or albumin promoter from rice (Wu et al., 1998, Plant and CellPhysiology 39: 885-889), a Vicia faba promoter from the legumin B4 andthe unknown seed protein gene from Vicia faba (Conrad et al., 1998,Journal of Plant Physiology 152: 708-711), a promoter from a seed oilbody protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, PlantPhysiology 102: 991-1000, the chlorella virus adenine methyltransferasegene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26:85-93), or the aldP gene promoter from rice (Kagaya et al., 1995,Molecular and General Genetics 248: 668-674), or a wound induciblepromoter such as the potato pin2 promoter (Xu et al., 1993, PlantMolecular Biology 22: 573-588). Likewise, the promoter may be inducibleby abiotic treatments such as temperature, drought or alterations insalinity or inducible by exogenously applied substances that activatethe promoter, e.g., ethanol, oestrogens, plant hormones like ethylene,abscisic acid, gibberellic acid, and/or heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of the polypeptide in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu at al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

Still further, the codon usage may be optimized for the plant species inquestion to improve expression (see Horvath et al. referred to above).

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19; 15-38), andit can also be used for transforming monocots, although othertransformation methods are more often used for these plants. Presently,the method of choice for generating transgenic monocots, supplementingthe Agrobacterium approach, is particle bombardment (microscopic gold ortungsten particles coated with the transforming DNA) of embryonic callior developing embryos (Christou, 1992, Plant Journal 2; 275-281;Shimamoto, 1994, Current Opinion Biotechnology 5; 158-162; Vasil et al.,1992, Bio/Technology 10; 667-674). An alternative method fortransformation of monocots is based on protoplast transformation asdescribed by Omirulleh et al., 1993, Plant Molecular Biology 21;415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using,e.g., co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having phytase activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Transgenic Animals

The present invention also relates to a transgenic, non-human animal andproducts or elements thereof, examples of which are body fluids such asmilk and blood, organs, flesh, and animal cells. Techniques forexpressing proteins, e.g., in mammalian cells, are known in the art,see, e.g., the handbook Protein Expression: A Practical Approach,Higgins and Hames (eds). Oxford University Press (1999), and the threeother handbooks in this series relating to Gene Transcription, RNAprocessing, and Post-translational Processing. Generally speaking, toprepare a transgenic animal, selected cells of a selected animal aretransformed with a nucleic acid sequence encoding a polypeptide havingphytase activity of the present invention so as to express and producethe polypeptide. The polypeptide may be recovered from the animal, e.g.,from the milk of female animals, or the polypeptide may be expressed tothe benefit of the animal itself, e.g., to assist the animal'sdigestion. Examples of animals are mentioned below in the section headedAnimal Feed.

To produce a transgenic animal with a view to recovering the polypeptidefrom the milk of the animal, a gene encoding the polypeptide may beinserted into the fertilized eggs of an animal in question, e.g., by useof a transgene expression vector which comprises a suitable milk proteinpromoter, and the gene encoding the polypeptide. The transgeneexpression vector is microinjected, into fertilized eggs, and preferablypermanently integrated into the chromosome. Once the egg begins to growand divide, the potential embryo is implanted into a surrogate mother,and animals carrying the transgene are identified. The resulting animalcan then be multiplied by conventional breeding. The polypeptide may bepurified from the animal's milk, see, e.g., Meade et al., 1999,Expression of recombinant proteins in the milk of transgenic animals,Gene expression systems: Using nature for the art of expression,Fernandez and Hoeffler (eds.), Academic Press.

In the alternative, in order to produce a transgenic non-human animalthat carries in the genome of its somatic and/or germ cells a nucleicacid sequence including a heterologous transgene construct including atransgene encoding the polypeptide, the transgene may be operably linkedto a first regulatory sequence for salivary gland specific expression ofthe polypeptide, as disclosed in WO 00/064247.

Compositions and Uses

In still further aspects, the present invention relates to compositionscomprising a polypeptide of the present invention, as well as methods ofusing these.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of granulates or microgranulates. The polypeptide to be included inthe composition may be stabilized in accordance with methods known inthe art.

The phytase of the invention can be used for degradation, in anyindustrial context, of, for example, phytate, phytic acid, and/or themono-, di-, tri-, tetra- and/or penta-phosphates of myo-inositol. It iswell known that the phosphate moieties of these compounds chelatesdivalent and trivalent cations such as metal ions, inter alia, thenutritionally essential ions of calcium, iron, zinc and magnesium aswell as the trace minerals manganese, copper and molybdenum. Besides,the phytic acid also to a certain extent binds proteins by electrostaticinteraction.

Accordingly, preferred uses of the polypeptides of the invention are inanimal feed preparations (including human food) or in additives for suchpreparations.

In a particular embodiment, the polypeptide of the invention can be usedfor improving the nutritional value of an animal feed. Non-limitingexamples of improving the nutritional value of animal feed (includinghuman food), are: Improving feed digestibility; promoting growth of theanimal; improving feed utilization; improving bio-availability ofproteins; increasing the level of digestible phosphate; improving therelease and/or degradation of phytate; improving bio-availability oftrace minerals; improving bio-availability of macro minerals;eliminating the need for adding supplemental phosphate, trace minerals,and/or macro minerals; and/or improving egg shell quality. Thenutritional value of the feed is therefore increased, and the growthrate and/or weight gain and/or feed conversion (i.e. the weight ofingested feed relative to weight gain) of the animal may be improved.

Furthermore, the polypeptide of the invention can be used for reducingphytate level of manure.

Animals, Animal Feed, and Animal Feed Additives

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants. Ruminant animals include,for example, animals such as sheep, goat, and cattle, e.g., cow such asbeef cattle and dairy cows. In a particular embodiment, the animal is anon-ruminant animal. Non-ruminant animals include mono-gastric animalse.g., pig or swine (including, but not limited to, piglets, growingpigs, and sows); poultry such as turkeys, ducks and chickens (includingbut not limited to broiler chicks, layers); fish (including but notlimited to salmon, trout, tilapia, catfish and carp), and crustaceans(including but not limited to shrimp and prawn).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the polypeptide can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the polypeptide, in the form in which it isadded to the feed, or when being included in a feed additive, issubstantially pure. In a particular embodiment it is well-defined. Theterm “well-defined” means that the phytase preparation is at least 50%pure as determined by Size-exclusion chromatography (see Example 12 ofWO 01/58275). In other particular embodiments the phytase preparation isat least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure asdetermined by this method.

A substantially pure, and/or well-defined polypeptide preparation isadvantageous. For instance, it is much easier to dose correctly to thefeed a polypeptide that is essentially free from interfering orcontaminating other polypeptides. The term dose correctly refers inparticular to the objective of obtaining consistent and constantresults, and the capability of optimising dosage based upon the desiredeffect.

For the use in animal feed, however, the phytase polypeptide of theinvention need not be that pure; it may, e.g., include otherpolypeptides, in which case it could be termed a phytase preparation.

The phytase preparation can be (a) added directly to the feed (or useddirectly in a treatment process of proteins), or (b) it can be used inthe production of one or more intermediate compositions such as feedadditives or premixes that is subsequently added to the feed (or used ina treatment process). The degree of purity described above refers to thepurity of the original polypeptide preparation, whether used accordingto (a) or (b) above.

Polypeptide preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the polypeptide is produced by traditionalfermentation methods.

Such polypeptide preparation may of course be mixed with otherpolypeptides.

The polypeptide can be added to the feed in any form, be it as arelatively pure polypeptide, or in admixture with other componentsintended for addition to animal feed, i.e., in the form of animal feedadditives, such as the so-called pre-mixes for animal feed.

In a further aspect the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives,e.g., premixes.

Apart from the polypeptide of the invention, the animal feed additivesof the invention contain at least one fat-soluble vitamin, and/or atleast one water soluble vitamin, and/or at least one trace mineral. Thefeed additive may also contain at least one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, e.g.,carotenoids such as beta-carotene, astaxanthin, and lutein; aromacompounds; stabilizers; antimicrobial peptides; polyunsaturated fattyacids; reactive oxygen generating species, and/or at least one otherpolypeptide selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26);phosphatase (EC 3.1.3.1; EC 3.1.3.2; EC 3.1.3.39); xylanase (EC3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22);protease (EC 3.4.-.-), phospholipase A1 (EC 3.1.1.32); phospholipase A2(EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3);phospholipase D (EC 3.1.4.4); amylase such as, for example,alpha-amylase (EC 3.2.1.1); and/or beta-glucanase (EC 3.2.1.4 or EC3.2.1.6).

In a particular embodiment these other polypeptides are well-defined (asdefined above for phytase preparations).

The phytase of the invention may also be combined with other phytases,for example ascomycete phytases such as Aspergillus phytases, forexample derived from Aspergillus awamoril, Aspergillus ficuum, orAspergillus niger, or basidiomycete phytases, for example derived fromAgrocybe pediades, Paxillus involutus, Peniophora lycii, or Trametespubescens; or derivatives, fragments or variants thereof which havephytase activity.

Thus in preferred embodiments of the use in animal feed of theinvention, and in preferred embodiments of the animal feed additive andthe animal feed of the invention, the phytase of the invention iscombined with such phytases.

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin Protegrin-1, Thanatin, Defensin, Lactoferrin, Lactoferricin,and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, andStatins, including the compounds and polypeptides disclosed in WO03/044049 and WO 03/048148, as well as variants or fragments of theabove that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and polypeptides such as anoxidase, an oxygenase or a syntethase.

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with a polypeptide of the invention, isan animal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g., vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1 vitamin B2, vitamin B6, niacin, folic acid and panthothenate,e.g., Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterised as indicated in Table B of WO01/58275, columns 2-3. Fish diets can be characterised as indicated incolumn 4 of this Table B. Furthermore such fish diets usually have acrude fat content of 200-310 g/kg.

WO 01/58275 corresponds to U.S. Ser. No. 09/779,334 which is herebyincorporated by reference.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least onepolypeptide as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolizable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R, 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e., Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolizable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen bv, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one protein. The protein may be an animal protein,such as meat and bone meal, and/or fish meal; or it may be a vegetableprotein. The term vegetable proteins as used herein refers to anycompound, composition, preparation or mixture that includes at least oneprotein derived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.,beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, sunflowerseed, cotton seed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, triticale, and sorghum.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats, and/or 0-40% soybeanmeal, and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

Animal diets can, e.g., be manufactured as mash feed (non pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question.Polypeptides can be added as solid or liquid polypeptide formulations.For example, a solid polypeptide formulation is typically added beforeor during the mixing step; and a liquid polypeptide preparation istypically added after the pelleting step. The polypeptide may also beincorporated in a feed additive or premix.

The final polypeptide concentration in the diet is within the range of0.01-200 mg polypeptide protein per kg diet, for example in the range of5-30 mg polypeptide protein per kg animal diet.

The phytase of the invention should of course be applied in an effectiveamount, i.e., in an amount adequate for improving solubilisation and/orimproving nutritional value of feed. It is at present contemplated thatthe polypeptide is administered in one or more of the following amounts(dosage ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 10-100;0.05-50; or 0.10-10—all these ranges being in mg phytase polypeptideprotein per kg feed (ppm).

For determining mg phytase polypeptide protein per kg feed, the phytaseis purified from the feed composition, and the specific activity of thepurified phytase is determined using a relevant assay. The phytaseactivity of the feed composition as such is also determined using thesame assay, and on the basis of these two determinations, the dosage inmg phytase protein per kg feed is calculated.

The same principles apply for determining mg phytase polypeptide proteinin feed additives. Of course, if a sample is available of the phytaseused for preparing the feed additive or the feed, the specific activityis determined from this sample (no need to purify the phytase from thefeed composition or the additive).

Methods for Producing Fermentation Products and Co-Products

Yet another aspect of the present invention relates to the methods forproducing a fermentation product/co-product, such as, e.g., ethanol,beer, wine, distillers dried grains (DDG), wherein the fermentation iscarried out in the presence of a phytase of the present invention.Examples of fermentation processes include, for example, the processesdescribed in WO 01/62947. Fermentation is carried out using a fermentingmicroorganism, such as, yeast.

In a particular embodiment, the present invention provides methods forproducing ethanol, comprising fermenting (using a fermentingmicroorganism, such as yeast) a carbohydrate containing material (e.g.,starch) in the presence of a phytase of the present invention.

In another embodiment, the present invention provides methods forproducing ethanol comprising hydrolyzing starch, e.g., by a liquefactionand/or saccharification process, a raw starch hydrolysis process,fermenting the resulting starch in the presence of a phytase of thepresent invention, and producing ethanol.

The phytase may be added to the fermentation process at any suitablestage and in any suitable composition, including atone or in combinationwith other enzymes, such as, one or more alpha-amylases, cellulases,glucoamylases, and proteases.

In another embodiment, the present invention provides methods forproducing ethanol comprising hydrolyzing biomass, and fermenting theresulting biomass in the presence of a phytase of the present invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Example 1 Cloning and Expression of Buttiauxella Phytases

A multiple alignment was made of the following histidine acidphosphatases (HAP): appA Escherichia coli (SPTREMBL:Q8GN88), Citrobactergillenii DSM 13694 phytase (geneseqp:aeh04533), Citrobacter amalonaticusATCC 25407 phytase (geneseqp:aeh04535), Citrobacter braakii phytase(geneseqp:aeh04827), and ypo1648 Yersinia pestis CO92 (SPTREMBL:Q8ZFP6).Two degenerate oligonucleotide primers were designed on the basis ofconsensus sequences:

2123: 5′-CATGGTGTGCGNGCNCCNAGNAA-3′ (SEQ ID NO: 9, forward primer) 2065:5′-CCCACCAGGNGGNGTRTTRTCNGGYTG-3′ (SEQ ID NO 10, reverse primer),wherein Y designates T or C, R designates A or G, and N designates A, C,G or T.

The primers were used for PCR screening of a number of bacterial speciesat annealing temperatures between 40 and 50° C. but typically as touchdown program starting with 50° C. and then reducing the annealingtemperature with 1° C. for each cycle over the next 10 cycles beforeconducting standard PCR.

A partial phytase gene in the form of an approximately 950 bp PCRfragment was identified in Buttiauxella agrestis DSM 18932.

The PCR fragment was isolated from agarose gel and the fragment wassequenced using the same PCR primers as those with which the fragmentwas generated. By translation of the nucleotide sequence, it wasconfirmed that the DNA fragment was part of a HAP phytase gene.

For obtaining the full length nucleotide sequence of the gene, the DNAWalking SpeedUp Kit (DWSK-V102 from Seegene, Inc., 2nd Fl., MyungjiBldg., 142-21, Samsung-dong, Kangnam-gu, Seoul, 135-090, Korea) wasused, which is designed to capture unknown target sites. For thispurpose, 4 specific oligonucleotides were designed and used with thekit:

(SEQ ID NO: 11) 2138 TSP1N: 5′-ATTGCGGAGCAAGCCCTG-3′ (SEQ ID NO: 12)2139 TSP2N: 5′-TCGCCAGTTTTAAGCGNCGG-3′ (SEQ ID NO: 13) 2136 TSP1C:5′-TGGAATATGCGCAAGGGATG-3′ (SEQ ID NO: 14) 2137 TSP2C:5′-TGGGGGTCAGAGCAAGAGTGGG-3′

The full length nucleotide sequence encoding the phytase fromButtiauxella agrestis DSM 18932 is shown in the sequence listing as SEQID NO: 5, and the corresponding encoded amino acid sequence has SEQ IDNO: 6. The first 33 amino acids of SEQ ID NO: 6 (i.e., amino acids −33to −1) are a signal peptide, as predicted by Signal P V3.0 (seewww.cbs.dtu.dk/services/SignalP/).

The same multiple alignment described above was used for design of twoother degenerate oligonucleotide PCR primers:

315: 5′-CGCGTGGTGATTGTGTCCMGNCAYGGNGT-3′ (SEQ ID NO: 15, forward primer)316: 5′-CCAGGTTGGTATCATGGCCNGCDATRAA-3′ (SEQ ID NO: 16. reverse primer).wherein Y designates T or C, M designates A or C, N designates A, C, Gor T, and R designates A or G.

The primers were used for PCR screening of a number of bacterial speciesat the same conditions as described above.

Partial phytase genes in the form of approximately 900 bp PCR fragmentswere identified in Buttiauxella gaviniae DSM 18930 as well as inButtiauxella agrestis DSM 18931.

The two PCR fragments were isolated from agarose gel and sequenced usingprimers 1978 and 1979:

1978: 5′-CGCGTGGTGATTGTGTCC-3′ (SEQ ID NO: 17) 1979:5′-CCAGGTTGGTATCATGGCC-3′ (SEQ ID NO: 18)

By translation of the nucleotide sequence, it was confirmed that bothDNA fragments were PCR amplified from HAP phytase genes.

Four new primers were designed for each gene for obtaining the fulllength nucleotide sequence of the gene according to the instructionsgiven in the DNA Walking SpeedUp Kit (DWSK-V102 from Seegene, Inc., 2ndFl., Myungji Bldg., 142-21, Samsung-dong, Kangnam-gu, Seoul, 135-090,Korea).

The four specific oligonucleotide PCR primers designed for theButtiauxella gaviniae DSM 18930 phytase gene were:

(SEQ ID NO: 19) 2029 TSP1N: 5′-AAGCTTCGCCAGTTTTAAGCG-3′ (SEQ ID NO: 20)2030 TSP2N: 5′-TTGAGTTTGGTGTGGGGCAACTG-3′ (SEQ ID NO: 21) 2031 TSP1C:5′-TGGCAACAAAGTCGCTCTCG-3′ (SEQ ID NO: 22) 2032 TSP2C:5′-TCCTGCTGGAATATGCGCAAGG-3′

The four specific oligonucleotide PCR primers designed for theButtiauxella agrestis DSM 18931 phytase gene were:

(SEQ ID NO: 23) 2017 TSP1N: 5′-TTCGCCCGTTTTAAGCGTG-3′ (SEQ ID NO: 24)2018 TSP1C: 5′-ACTGCCCTGCGATAAAATGCCC-3′ (SEQ ID NO: 25) 2019 TSP2N:5′-TTTCCTGCTGGAATATGCGC-3′ (SEQ ID NO: 26) 2020 TSP2C:5′-TTGATGGCGCGCACACCTTAC-3′

The full length nucleotide sequence encoding the phytase fromButtiauxella gaviniae DSM 18930 is shown in the sequence listing as SEQID NO: 1, and the corresponding encoded amino acid sequence has SEQ IDNO: 2. The first 33 amino acids of SEQ ID NO: 2 (i.e., amino acids −33to −1) are expected to be a signal peptide (predicted by Signal P V3.0,see www.cbs.dtu.dk/services/SignalP/).

The full length nucleotide sequence encoding the mature phytase, as wellas a partial sequence coding for the signal peptide from Buttiauxellaagrestis DSM 18931 is shown in the sequence listing as SEQ ID NO: 3. Thecorresponding encoded amino acid sequence has SEQ ID NO: 4. The first 9amino acids of SEQ ID NO: 4 (i.e., amino acids −9 to −1) are expected tobe a part of the signal peptide.

The three phytase genes were expressed in Bacillus subtilis as follows:

The signal peptide encoding sequence of SEQ ID NO: 7 (encoding thesignal peptide of SEQ ID NO: 8 and derived from a Bacillus licheniformisprotease) was fused by PCR in frame to the gene encoding the maturephytase from each of the three phytases in turn.

The DNA coding for the resulting coding sequence was integrated byhomologous recombination on the Bacillus subtilis host cell genome. Thegene constructs were expressed under the control of a triple promotersystem (as described in WO 99/43835), consisting of the promoters fromBacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis alpha-amylase gene (amyL), and the Bacillus thuringiensiscryIIIA promoter including stabilizing sequence. The gene coding forChloramphenicol acetyl-transferase was used as marker as described in,e.g., Diderichsen et al., 1993, A useful cloning vector for Bacillussubtilis, Plasmid 30: 312.

For each of the three constructs, chloramphenicol resistanttransformants were cultured in PS-1 medium (10% sucrose, 4% soybeanflour, 1% Na₃PO₄-12H₂O, 0.5% CaCO₃, and 0.01% pluronic acid), whileshaking at 250 rpm at 30° C. After 2-5 days of incubation thesupernatants were removed and the phytase activity was identified byapplying 20 microliters of the supernatant into 4 mm diameter holespunched out in 1% LSB-agarose plates containing 0.1 M Sodium acetate pH4.5 and 0.1% Inositol hexaphosphoric acid. The plates were left overnight at 37° C. and a buffer consisting of 0.25 M CaCl₂ and 500 mM MES(adjusted to pH 6.5 with 4 N NaOH) were poured over the plates. Theplates were left at room temperature for 1 h and the inositolphosphatephosphatase, or phytase, activity was then identified as a clear zone.

Several phytase positive transformants for each of the three constructswere analyzed by DNA sequencing to ensure the correct DNA sequence ofthe constructs. One correct clone was selected for each of the threeconstructs.

Example 2 Fermentation and Purification of Buttiauxella Phytases

The strain of Bacillus subtilis harboring the Buttiauxella agrestis DSM18932 phytase construct and capable of expressing the phytase having SEQID NO: 6 (mature part) was cultivated at 30° C. and with 250 rpm for 6days in SK-1M medium (Sodium Caseinate (Miprodan 30 from Arla) 40 g,Maltodextrin 01 (Glucidex 6, catalogue no. 332203 from Roquette), 200 g,Soybean Meal 50 g, Dowfax 63N10 (a non-ionic surfactant from Dow) 0.1ml, tap water up to 1000 ml, CaCO₃ tablet 0.5 g/100 ml).

The strain of Bacillus subtilis harboring the Buttiauxella agrestis DSM18931 phytase construct and capable of expressing the phytase having SEQID NO: 4 (mature part) was cultivated at 30° C. and with 250 rpm for 5days in the PS-1 medium which is described in Example 1

The strain of Bacillus subtilis harboring the Buttiauxella gavniae DSM18930 phytase construct and capable of expressing the phytase having SEQID NO: 2 (mature part) was cultivated at 30° C. and with 250 rpm for 5days in the PS-1 medium which is described in Example 1.

The fermentation supernatant with the phytase of SEQ ID NO: 2 was firstcentrifuged at 7200 rpm and 5° C. for 2 hours and filtered through aFast PES Bottle top filter with a 0.22 micro-m cut-off. Next, thefiltered supernatant was pre-treated as follows.

The sample solution was washed with water and concentrated using anultrafiltration unit (Filtron, from Filtron Technology Corporation)equipped with a 10 kDa cut-off ultrafiltration membrane.

Then pH was adjusted to 5.0 with 6 M HCl, which caused a minorprecipitation. This was removed by centrifugation of the sample at 7200rpm for 20 minutes at 5° C. The supernatant, containing the phytase, wasadded 2.5 ml 1 M sodium acetate pH 5.0 and filtered through a Fast PESbottle top filter with a 0.22 micro-m cut-off. After this the pH of thesolution was measured to 5.0 and the conductivity was found to be 1.8mS/cm. After pretreatment the phytase was purified by chromatography onSP Sepharose, approximately 85 ml in a XK6 column, using as buffer A 20mM sodium acetate pH 5.0, and as buffer B 20 mM sodium acetate+1 M NaClpH 5.0.

The fractions from the column were analyzed for phytase activity andfractions with activity were pooled.

The phytase of SEQ ID NO: 4 was purified essentially as described above,except that 10% acetic acid was used to adjust the pH. Again someprecipitation was observed and this was removed by centrifugation. ThepH of the solution was measured to 5.0, while the conductivity was foundto be approximately 1.2 mS/cm before the column chromatography step.

The fermentation supernatant with the phytase of SEQ ID NO: 6 was firstcentrifuged at 7200 rpm and 5° C. for one hour and filtered through asandwich of four Whatman glass microfibre filters (2.7, 1.6, 1.2 and 0.7micrometer). Following this the solution was filtered through aSeitz-EKS depth filter using pressure. The solution was added solidammonium sulfate giving a final concentration of 1.5 M and the pH wasadjusted to 6.0 using 6 M HCl.

The phytase-containing solution was applied to a butyl-sepharose column,approximately 50 ml in a XK26 column, using as buffer A 25 mMbis-tris+1.5 M ammonium sulfate pH 6.0, and as buffer B 25 mM bis-trispH 6.0. However, the enzyme did not bind to the column and almost allactivity was found in the flow-through and wash fractions. Theflow-through and wash fractions were combined and solid ammonium sulfatewas added to about 80% saturation. The solution was left overnight at 5°C. in order to complete precipitation. The precipitate was isolated (noactivity was found in the supernatant) and dissolved in Milli-Q water.This solution was dialyzed against Milli-Q water and pH was adjusted to4.5.

Following this the phytase was purified by chromatography on SSepharose, approximately 150 ml in a XK26 column, using as buffer A 50mM sodium acetate pH 4.5, and as buffer B 50 mM sodium acetate+1 M NaClpH 4.5.

The fractions from the column were analyzed for phytase activity andfractions with activity were pooled. Finally, the fractions with phytaseactivity were concentrated using an Amicon ultra-15 filtering devicewith a 30 kDa cut-off membrane.

The molecular weight, as estimated from SDS-PAGE, was approximately 40kDa for all three phytases and the purity was in all cases>95%.

Example 3 Phytase Activity Assay

Phytase activity may suitably be determined by the following assay:

75 microliters phytase-containing enzyme solution, appropriately dilutedin 0.25 M sodium acetate, 0.005% (w/v) Tween-20, pH 5.5, is dispensed ina microtiter plate well, e.g., NUNC 269620, and 75 microliters substrateis added (prepared by dissolving 100 mg sodium phytate from rice(Aldrich Cat. No. 274321) in 10 ml 0.25 M sodium acetate buffer, pH5.5). The plate is sealed and incubated 15 nm, shaken with 750 rpm at37° C. After incubation, 75 microliters stop reagent is added (the stopreagent being prepared by mixing 10 ml molybdate solution (10% (w/v)ammonium hepta-molybdate in 0.25% (w/v) ammonia solution), 10 mlammonium vanadate (0.24% commercial product from Bie&Berntsen, Cat. No.LAB17650), and 20 ml 21.7% (w/v) nitric acid), and the absorbance at 405nm is measured in a microtiter plate spectrophotometer. The phytaseactivity is expressed in the unit of FYT, one FYT being the amount ofenzyme that liberates 1 micromole inorganic orthophosphate per minuteunder the conditions above. An absolute value for the measured phytaseactivity may be obtained by reference to a standard curve prepared fromappropriate dilutions of inorganic phosphate, or by reference to astandard curve made from dilutions of a phytase enzyme preparation withknown activity (such standard enzyme preparation with a known activityis available on request from Novozymes A/S, Krogshoejvej 36, DK-2880Bagsvaerd).

Example 4 Specific Activity

The specific activity of the phytases from Buttiauxella gaviniae DSM18930, Buttiauxella agrestis DSM 18931, and Buttiauxella agrestis DSM18932 (having the amino acid sequences of the mature parts of SEQ ID NO:2, 4 and 6, respectively) was determined in sodium acetate buffer, pH5.5. The phytases were highly purified as described in Example 2, i.e.,only one component was identified on an SDS polyacrylamide get.

The protein concentration was determined by amino acid analysis asfollows: An aliquot of the sample was hydrolyzed in 6 M HCl, 0.1% phenolfor 16 h at 110° C. in an evacuated glass tube. The resulting aminoacids were quantified using an Applied Biosystems 420A amino acidanalysis system operated according to the manufacturer's instructions.From the amounts of the amino acids the total mass—and thus also theconcentration—of protein in the hydrolyzed aliquot was calculated.

The phytase activity was determined in the units of FYT as described inExample 3, and the specific activity was calculated as the phytaseactivity measured in FYT units per mg phytase enzyme protein.

The resulting specific activities for the three phytases are shown inTable 2. The specific activity was determined on sodium phytate at pH5.5 and 37° C.

TABLE 2 Specific activity Phytase (FYT/mg protein) SEQ ID NO: 2 700 SEQID NO: 4 800 SEQ ID NO: 6 650

The wildtype (wt) phytase from Buttiauxella P1-29 described in WO2006/043178 has a specific activity at pH 3.5 and 37° C. of about 300U/mg (see Example 10 of this WO publication). According to Example 2thereof, the ratio of activities at pH 3.5 and pH 5.5 is about 1.3,while according to FIG. 2 (pH activity profile of the purified phytase)this ratio is rather 1.5. In any event, when the specific activity at pH3.5 is about 300 U/mg, the specific activity at pH 5.5 is not higherthan 300/1.3=231 U/mg (maybe it is rather 300/1.5=200 U/mg).

WO 2006/043178 also discloses a variant phytase with a specific activityat pH 4.0 and 37° C. which is 115% of the specific activity of the wt.Using again FIG. 2, the specific activity of the wt at pH 4.0 is nothigher than 80/60×300 U/mg=400 U/mg. The specific activity at pH 4.0 ofthe variant is then 1.15×400 U/mg=460 U/mg, while at pH 5.5 the specificactivity of this variant is approximately 40/80×460 U/mg=230 U/mg (infact, 230 is precisely 115% of the 200 U/mg of the wildtype at pH 5.5,as calculated above).

Apart from the pH-difference, which has been taken into account above,the assay conditions of WO 2006/043178 are, for all practical purposes,identical to those of the present invention, except possibly for thepresence in WO 2006/043178 of 0.8 mM CaCl₂ in the reaction mixture.However, we have tested the activity of the phytases of SEQ ID NOs: 2, 4and 6 in the assay of Example 3 including even more Ca²⁺ (viz., 1 mMCaCl₂) but found no difference.

Accordingly, the phytases of the present invention have a very muchimproved specific activity at pH 5.5 as compared to the phytasesdisclosed in WO 2006/043178. In fact, the specific activity of thephytases of the invention is improved in the whole range of pH 3.5-5.5.This is apparent from Example 6 below, which shows that for the phytasesof the invention the activity in this range is at least as high as theactivity at pH 5.5.

Example 5 pI

The isoelectric point, pI, for the three phytases was determined usingisoelectric focusing gels (Novex pH 310 IEF gel from Invitrogen, catalognumber EC6655A2) run as described by the manufacturer. The pI for theButtiauxella agrestis DSM 18931 and DSM 18932 phytases (SEQ ID NOs: 4and 6) is 7.4, while the pI for the Buttiauxella gaviniae DSM 18930 (SEQID NO: 2) phytase is 7.6.

Example 6 pH Profile

The pH profile (phytase activity as a function of pH) of the threephytases was determined in the pH range of 2.0 to 7.5 as described inExample 3, except that a buffer cocktail (50 mM glycine, 50 mM aceticacid and 50 mM Bis-Tris(Bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methan)) was used insteadof the 0.25 M sodium acetate pH 5.5 buffer.

The results are shown in Table 3 below, relative to the value at theoptimum pH for each phytase (pH 4.5).

TABLE 3 Relative phytase activity versus pH Buttiauxella gaviniaeButtiauxella agrestis Buttiauxella noackiea DSM 18930 DSM 18931 DSM18932 pH (SEQ ID NO: 2) (SEQ ID NO: 4) (SEQ ID NO: 6) 2.0 37 41 40 2.546 49 49 3.0 66 65 66 3.5 83 86 78 4.0 96 99 99 4.5 100 100 100 5.0 9291 83 5.5 70 70 69 6.0 38 43 39 6.5 11 14 14 7.0 −3 1 1 7.5 −5 −2 0

As compared to the pH activity profile of the wildtype (wt) phytase fromButtiauxella P1-29 described in WO 2006/043178 (see FIG. 2 thereof), thephytases of the invention appear to have a higher relative activity atlow as well as high pH-values (at pH 2.0 and pH 6.0 the activity of thephytases of the present invention is twice as high, and eight times ashigh, respectively, as compared to the Buttiauxella P1-29 wt phytase).

Example 7 pH Stability

The pH stability of the purified phytases of SEQ ID NOs: 2 and 4 at 40°C. was determined by measuring residual phytase activity afterincubation at 40° C. and at various pH values for 1.5 and 24 hours. Thephytases were incubated in 0.1 M glycine, 0.1 M acetic acid, 0.1 MBis-Tris, adjusted to the desired pH. Samples of the respectiveincubation mixtures were withdrawn after 0, 1.5 and 24 hours, the pH ofthe samples was adjusted to 5.5 by dilution in 0.25 M sodium acetate,0.005% (w/v) Tween20, pH 5.5), and the residual activity at pH 5.5 wasdetermined using the method described in Example 3. The results,normalized to the activity found at 0 hours, are shown in Table 4 below.

TABLE 4 pH stability at 40° C. Buttiauxella Buttiauxella Buttiauxellagaviniae Buttiauxella gaviniae agrestis (SEQ ID agrestis (SEQ ID (SEQ IDpH NO: 2) (SEQ ID NO: 4) pH NO: 2) NO: 4) 1.5 hours 24 hours 2.0 77 832.0 51 56 3.0 74 85 3.0 66 66 4.0 81 95 4.0 77 88 5.0 80 68 5.0 78 676.0 82 67 6.0 76 67 7.0 79 75 7.0 76 73 8.0 80 74 8.0 77 70

Both phytases are very stable for 1.5 hours in the entire range of pH2.0-8.0, whereas when incubated for 24 hours a certain loss of activityis observed at the lower pH values (pH 2.0-3.0).

Example 8 Temperature Profile

The temperature profile (phytase activity as a function of temperature)of the three phytases was determined in the temperature range of 20-90°C. essentially as described in Example 3, however, the enzymaticreactions (100 microliters phytase-containing enzyme solution+100microliters substrate) were performed in PCR tubes instead of microtiterplates. After a 15 minute reaction period at desired temperature thetubes were cooled to 20° C. for 20 seconds and 150 microliters of thereaction mixture was transferred to a microtitter plate. 75 microlitersstop reagent was added and the absorbance at 405 nm was measured in amicrotiter plate spectrophotometer.

The temperature profiles for the phytases of SEQ ID NOs: 2 and 4 weredetermined at pH 4.0 (0.25 M sodium acetate), whereas the temperatureprofile for the phytase of SEQ ID NO: 6 was determined at pH 5.5 (0.25 Msodium acetate).

The results are shown in Table 5 below, for each phytase relative to theactivity at the optimum temperature.

TABLE 5 Temperature profile at pH 4.0/5.5 Temperature (° C.) 20 30 40 5060 70 80 90 SEQ ID NO: 2 21 36 43 75 100 23 9 6 SEQ ID NO: 4 24 40 54 82100 17 8 7 SEQ ID NO: 6 22 32 50 74 100 14 8 3

All phytases appear to have an optimum temperature of about 60° C., arefairly active in the temperature range of 30-60° C., and also show adecent activity at 20° C. as well as at 70° C. In the range of 80-90° C.the activity is insignificant.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with DSMZ (Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH). Inhoffenstraβe 7 B, D-38124 Braunschweig, Germany,and given the following accession numbers:

Deposit Accession Number Date of Deposit Buttiauxella gaviniae DSM 1893015-JAN-2007 Buttiauxella agrestis DSM 18931 15-JAN-2007 Buttiauxellaagrestis DSM 18932 15-JAN-2007

The strains have been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subject applicationor its progeny are filed. However it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The above strains were isolated from samples collected in Denmark in2005.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-37. (canceled)
 38. A polypeptide having phytase activity and having anamino acid sequence which a) has at least 70% identity to amino acids1-413 of SEQ ID NO: 2, amino acids 1-413 of SEQ ID NO: 4, and/or aminoacids 1-413 of SEQ ID NO: 6 when aligned to the respective amino acidsequence using the Needle program with the BLOSUM62 substitution matrix,a gap opening penalty of 10.0, and a gap extension penalty of 0.5; andb) comprises at least one of the following amino acids at the positionindicated: 109Q, 111G, 119N, 120L, and/or 121E, when aligned asdescribed in a) to amino acids 1-413 of SEQ ID NO: 2 and using an aminoacid residue numbering corresponding to amino acids 1-413 of SEQ ID NO:2.
 39. A polypeptide having phytase activity and having an amino acidsequence which a) has at least 78% identity to amino acids 1-413 of SEQID NO: 2, amino acids 1-413 of SEQ ID NO: 4, and/or amino acids 1-413 ofSEQ ID NO: 6 when aligned to the respective amino acid sequence usingthe Needle program with the BLOSUM62 substitution matrix, a gap openingpenalty of 10.0, and a gap extension penalty of 0.5; and b) comprises atleast one of the following amino acids at the position indicated: 1S,10I, 38S, 66E, 71K, 81A, 109Q, 111G, 119N, 120L, 121E, 141R, 142L, 152M,155E, 193Q, 214V, 239K, 245D, 248E, 255A,T, 268A,T, 277T, 283D,E, 285K,287D, 288A,V, 293G, 296S, 303L, 314A, 337I, 345A, 350I, 364A, 371K,372E, 396P, 399K, 406E, and/or 413P, when aligned as described in a) toamino acids 1-413 of SEQ ID NO: 2 and using an amino acid residuenumbering corresponding to amino acids 1-413 of SEQ ID NO:
 2. 40. Thepolypeptide of claim 38, the amino acid sequence of which comprises atleast one of the following amino acids at the position indicated: 26E,37Y, 89T, 92E, 134I,V, 160R, 164F, 171I, 176K, 178P, 188N, 190E, 192G,207E,T, 209S, 211C, 235V, 248L, 256H,Y, 261E, 270K, 303F, and/or 318D.41. A composition comprising at least one phytase of claim 38, and (a)at least one fat soluble vitamin; (b) at least one water solublevitamin, and/or (c) at least one trace mineral.
 42. The composition ofclaim 41 further comprising at least one enzyme selected from thefollowing group of enzymes: amylase, phytase, phosphatase, xylanase,galactanase, alpha-galactosidase, protease, phospholipase, and/orbeta-glucanase.
 43. The composition of claim 41 which is an animal feedadditive.
 44. An animal feed composition having a crude protein contentof 50 to 800 g/kg and comprising the phytase of claim
 38. 45. A methodfor improving the nutritional value of an animal feed, comprising addinga phytase of claim 38 to the feed.
 46. A process for reducing phytatelevels in animal manure comprising feeding an animal with an effectiveamount of the feed composition of claim
 44. 47. A method for thetreatment of vegetable proteins, comprising the step of adding a phytaseof claim 38 to the vegetable proteins.
 48. A method for producing afermentation product comprising fermenting a carbohydrate material inthe presence of a phytase of claim
 38. 49. A method for producingethanol comprising (a) fermenting a carbohydrate material in thepresence of a phytase of claim 38, and (b) producing ethanol.
 50. Apolynucleotide comprising a nucleotide sequence which encodes thepolypeptide of claim
 38. 51. A nucleic acid construct comprising thepolynucleotide of claim 50 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.
 52. A recombinant expression vector comprising the nucleic acidconstruct of claim
 51. 53. A recombinant host cell comprising thenucleic acid construct of claim
 51. 54. A method for producing thepolypeptide of claim 38 comprising (a) cultivating a host cellcomprising a nucleic acid construct comprising a nucleotide sequenceencoding the polypeptide under conditions conductive for production ofthe polypeptide; and (b) recovering the polypeptide.
 55. A transgenicplant, plant part or plant cell, which has been transformed with apolynucleotide encoding the polypeptide of claim
 50. 56. A transgenic,non-human animal, or products, or elements thereof, being capable ofexpressing a phytase of claim 50.